RNA Assumes More Prominent Role

Hepatitis C virus RNA binds to 40S ribosomal subunits during viral infection. The start codon, depicted as a green, yellow, or red circle, is positioned in the mRNA binding cleft of the ribosome by the structure of the IRES pseudoknot domain. As this is a necessary step for viral protein synthesis, 2'OMe oligonucleotides were designed to disrupt the pseudoknot domain structure and effectively inhibited protein synthesis by the HCV IRES. [Jennifer Doudna, University of California, Berkeley]

RNA is beginning to take its place among the movers and shakers of biology. For decades, it was considered a background player in biology, a static carrier of genetic instructions from big daddy DNA. Now, however, RNA and its analysis are coming into the spotlight in an ever-expanding arena. New players bearing acronyms such as piRNA, siRNA, shRNA, and ncRNA are now regulars in scientific lexicons (and, perhaps more importantly, Wikipedia).

Emerging trends and new advances in the field are reviewed in this article, including revolutionary tools for transcriptomic analyses, deciphering how pathogens subvert immune systems, and new modeling methods to better delineate RNA structure/function.

Piwi-interacting RNAs (piRNAs) are a new chapter in the emerging story of RNA biology. These small, noncoding RNAs (ncRNA) were discovered from a search for ncRNAs that interacted with the Drosophila signaling protein called Piwi. Two classes of piRNAs have been discovered. One set is abundant in germ cells and a second set is present in somatic cells and has a role in regulation of mRNA from early embryos and gonads.

The recent discovery of piRNAs in the central nervous system (CNS) was a surprise, reported Kenneth S. Kosik, M.D., professor and director, Neuroscience Research Institute, University of California, Santa Barbara.

“We are interested in how synapses of the CNS are locally regulated,” he said. “Since their discovery in 2006, the roles of piRNAs have continued to expand. We performed deep sequencing as a nonbiased approach toward searching for any category of small RNAs in the hippocampus, a brain region commonly used in studies of plasticity and studies of local translation in neuronal dendrites, an important facet of plasticity. Among the small RNA sequences observed were a set of piRNAs.”

Dr. Kosik and colleagues prepared small RNA libraries made by extracting RNA from male mouse hippocampus tissue to capture a large number of RNAs.

“We found a set of small RNAs in the size range of 24–31 base pairs, which is characteristic of piRNAs and quite distinct from the smaller ~21 nt microRNAs. We searched the piRNA database and found annotated sequences that corresponded to those in our sample. Then we confirmed our results by co-immunoprecipitating the piRNAs with the protein they bind to in the mouse (termed MIWI).”

The scientists also validated the results by performing in situ hybridization for several of the piRNAs. “Interestingly, we found that one piRNA extended out to dendrites; it was not confined to the neuron body. The dendrite is a site for a great deal of control over RNA and protein synthesis. These results may help us understand the plasticity of the nervous system.

“While we don’t know if piRNAs will have a role here, it certainly opens up the doors to explore if they are players. Minimally these studies revealed that piRNAs should no longer be considered as limited to the germ line. It is becoming increasingly clear that we need to think more broadly about them.”

RNA Sequencing

RNA sequencing is helping to revolutionize the characterization of gene expression. But, can the technology unmask the transcriptome of poorly understood and elusive pathogens such as Campylobacter jejuni? Yes, said Andrew J. Grant, Ph.D., senior research associate in the department of veterinary medicine, University of Cambridge, U.K.

“C. jejuni is the most common cause of bacterial food-borne diseases in the developed world. Its genome of ~1.6 megabases was sequenced around a decade ago, but we still understand very little about the pathogen.

“Our lab wants to better understand the roles of individual genes and proteins involved in its colonization, survival, and virulence as well as the general biochemistry and physiology of the bug. We utilized functional genomic approaches to do this, including sequencing the entire transcriptome of C. jejuni. At the time we began our studies, RNA sequencing of prokaryotes was very limited.”

According to Dr. Grant, high-throughput RNA sequencing can provide an unprecedented means to perform functional genomics. “Recently the transcriptomes of several eukaryotes and prokaryotes have been profiled using direct high-throughput Illumina sequencing of cDNAs, a process known as RNA-seq. We utilized Illumina high-throughput DNA sequencing to study mRNA expression levels of C. jejuni and then compared that to protein expression data.”

The Illumina platform has the capacity to generate tens of millions to billions of reads per run. The data can be aligned across splice junctions to assist characterizing novel transcripts, gene fusions, and isoforms. The technology is more sensitive than DNA microarrays that can miss ncRNAs.

To perform the studies, Dr. Grant and colleagues first isolated total RNA, subtracted out the 16 and 23 rRNA, and then reverse transcribed the resulting RNA into a cDNA library that could be sequenced on an Illumina Genome Analyzer.

“Our studies demonstrated the efficacy of high-throughput sequencing for profiling mRNA expression levels and for identifying genes that are differentially expressed. The technology also allowed us to identify a number of novel nonannotated ncRNA genes.”

Dr. Grant’s group is now developing the RNA-seq technology for use in mice infected with Salmonella enterica. “We hope to track expression changes in specific tissues or cells during active infection. Profiling both the transcriptome and the proteome is a powerful way to get at the bigger picture of what happens during infection by pathogens.”

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